The transcriptional activator NrpA is crucial for inducing nitrogen fixation in Methanosarcina mazei Gö1 under nitrogen‐limited conditions

With the aim of unraveling their potential involvement in the regulation of nitrogen metabolism in Methanosarcina mazei strain Gö1, we characterized five genes that are differentially transcribed in response to changing nitrogen availability and encoding putative transcriptional regulators. Study of the respective mutant strains under nitrogen‐limited conditions revealed a growth delay for M. mazei MM 0444::pac and MM 1708::pac , and strongly reduced diazotrophic growth for MM 0872::pac , whereas the absence of MM 2441 or MM 2525 did not affect growth behaviour. Transcriptome analyses further demonstrated that only MM 1708  – encoding a Cxx CG zinc finger protein – plays a regulatory role in nitrogen metabolism, most likely by specifically enhancing transcription of the N 2 fixation ( nif ) operon under nitrogen‐limited conditions. In agreement with this, a palindromic binding motif was predicted in silico in the nifH promoter region, nine nucleotides upstream of the BRE box, and confirmed to bind purified maltose‐binding protein– MM 1708 by electromobility shift assays. As MM 1708 itself is under the control of the global nitrogen repressor NrpR, this adds a secondary level to the transcriptional regulation of the nif genes, and is most likely crucial for maximal nif induction under nitrogen‐limited conditions. This is in accordance with the finding that protein expression of NifH is highly reduced in the absence of MM 1708 under nitrogen‐limited conditions. On the basis of our findings, we hypothesize that, in M. mazei , nitrogen fixation is controlled by a hierarchical network of two transcriptional regulators, the global nitrogen repressor NrpR, and the newly identified activator NrpA ( MM 1708), thereby providing tight control of N 2 fixation.